The present invention relates to a method of locating insertion elements (IS elements) or transposons in coryneform bacteria, a positive selection system suitable for the above, the IS elements found in this manner and their use.
Insertion elements (IS elements) are DNA which range from approximately 0.6 to 1.8 kilobases (kb) long and which can jump (transpose) in procaryotic genomes within a replicon or from one replicon to another (Craig & Kleckner 1987, in Neidhardt et al., "Escherichia coli and Salmonella typhimurium", Cellular and Molecular Biology, pp. 1054-1074, ASM Press, Washington, D.C.). This can result either in a conservative transposition (i.e., an element changes its position) or in a replicative transposition (i.e., in which only a copy of the element integrates at the new insertion site whereas the original remains at the old position). A fusion of the donor molecule and of the acceptor molecule can occur in replicative transposition (replicon fusion). This intermediary stage of the transposition can then be disintegrated again by means of a recombination of the copies of the IS elements located at the fusion points. However, the replicon fusion can be retained if there is a suitable selection for it. IS elements themselves are not equipped with a selectable marker, in contrast to the closely related transposons.
IS elements usually code only for a single gene product, the so-called transposase. This is a recombination protein which is read by one or two open reading frames of the insertion element and which carries out the transposition by means of so-called illegitimate recombination (i.e., independent of the recombination system of the host organism) at the inversely repetitive ends of the element.
During the transposition of insertion elements into a bacterial gene, the latter is usually destroyed; thus, a mutation is produced (Craig & Kleckner 1987). In addition, a disconnection of genes located further to the rear can occur due to polar effects, the interruption of the transcription of an operon by means of the integration into a front gene.
Endogenic insertion elements can contribute to the genetic instability of a natural or recombinant microorganism. In addition to insertions of IS elements, deletions of bordering regions or other rearrangements of DNA are produced thereby. It is also known that insertion elements can exert a negative influence on the stability of plasmids, especially under production conditions (Kumar et al., Trends Biotech. (1991), volume 9, pages 279-284). Insertion elements have already been demonstrated in a number of various bacterial genera. In Gram-positive bacteria, insertion elements are known in particular from the genera Bacillus, Staphylococcus, Streptococcus, Lactobacillus and Streptomyces.
Insertion elements from coryneform bacteria, especially those producing amino acids, have not yet been described in the art.